The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.

An undefined property of L-type Ca2+ channels is believed to underlie the unique phenotype of hibernating hearts. Therefore, L-type Ca2+ channels in single cardiomyocytes isolated from hibernating versus awake ground-squirrels (Citellus undulatus) were compared using the perforated mode of the patch-clamp technique, and interpreted by way of a kinetic model of Ca2+ channel behavior based upon the concept of independence of the activation and inactivation processes. We find that, in hibernating ground-squirrels, the cardiac L-type Ca2+ current is lower in magnitude when compared to awake animals. Both in the awake or hibernating states, kinetics of L-type Ca2+ channels could be described by a d2f1(2)f2 model with an activation and two inactivation processes. The activation (or d) process relates to the movement of the gating charge. The slow (or f1) inactivation is associated with movement of gating charge and is current-dependent. The rapid (or f2) inactivation is a complex process which cannot be represented as a single-step conformational transition induced by the gating charge movement, and is regulated by beta-adrenoceptor stimulation. When compared to awake animals, the kinetic properties of Ca2+ channels from hibernating ground-squirrels differed in the following parameters: (1) pronounced shift (15-20 mV) toward depolarization in the normalized conductance of both inactivation components, and moderate shift in the activation component; (2) 1.5-2-fold greater time constants; and (3) two-fold greater activation gating charge. Thus, L-type Ca2+ channels apparently switch their phenotype during the hibernating transition. Stimulation of beta-adrenoceptors by isoproterenol, reversed the hibernating kinetic- (but not amplitude-) phenotype toward the awake type. Therefore, an aberrance in the beta-adrenergic system can not fully explain the observed changes in the L-type Ca2+ current. This suggests that during hibernation additional mechanisms may reduce the single Ca2+ channel-conductance and/or keep a fraction of the cardiac L-type Ca2+ channel population in a non-active state.

The action of taxifolin on the angiotensinconverting enzyme (ACE) and the formation of reactive oxygen and nitrogen species (ROS/RNS) in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor (N ω -nitro-L-arginine methyl ester (L-NAME)) or dexamethasone have been studied. The ACE activity in aorta sections was determined by measuring the hydrolysis of hippuryl-L-histidyl-L-leucine, and the ROS/ RNS production was measured by oxidation of dichlorodihydrofluorescein. It was shown that taxifolin at a dose of 30-100 μg/kg/day decreases the ACE activity in the aorta of aging rats and of rats treated with L-NAME or dexamethasone to the level of the ACE activity in young control rats. Taxifolin (100 μg/kg/day) was found to also reduce the amount of ROS/RNS in the aorta that increased as a result of L-NAME intake. L-NAME treatment increases the contribution of 5-lipoxygenase and NADPH oxidase to ROS/RNS production in the aorta, while taxifolin (100 μg/kg/day) decreases the contribution of these enzymes to the normal level.

We studied changes in ROS content in the aorta of Wistar rats at early terms after irradiation in doses equal to single fraction used in tumor radiotherapy and the effects of taxifolin and fucoidin, blockers of leukocyte adhesion to endothelium, on ROS content. Male rats were exposed to X-rays (200 kW) in doses of 1-7.5 Gy. ROS production in aorta segments was measured in 1-48 h after irradiation by dichlorodihydrofluorescein oxidation. The content of ROS in the aorta of rats exposed to radiation in doses of 1-2.5 Gy increased in 1-24 h after irradiation, the peak ROS content was found in 2 h after irradiation. Taxifolin (100 μg/kg dihydroquercetin once a day with drinking water) and fucoidin (10 mg/kg, i.v.) abolished ROS accumulation. The content of ROS in rat aorta increased in 1-24 h after irradiation in doses used for tumor radiotherapy and this increase can be determined by leukocyte adhesion to the endothelium.

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH(2)) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH(2)-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.